All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Inflammation plays a crucial role in defense against pathogen invaders as well as in healing and recovery processes following various types of injury. However, the magnitude and duration of inflammatory responses have to be tightly regulated, because excessive inflammatory reactions can be detrimental, leading to autoimmune diseases, neurodegeneration, sepsis, trauma and other pathological conditions. It has long been recognized that regulation of inflammatory reactions is mediated both by immune responses (particularly the secretion of anti-inflammatory cytokines) and by neuroendocrine factors, particularly the activation of the pituitary-adrenal axis and the secretion of glucocorticoids. Recently it became evident that neural mechanisms are also involved in limiting inflammatory responses. In particular, it was found that cholinergic neurons inhibit acute inflammation, providing a rapid, localized, and adaptive anti-inflammatory reflex system (Tracy, 2002). In the periphery, acetylcholine (ACh) is mainly released by the efferent vagus nerve. It significantly attenuates the production of the pro-inflammatory cytokines TNFα, interleukin-1β (IL-1β), IL-6 and IL-18, but not the anti-inflammatory cytokine IL-10 [Tracey, K. J. (2002) Nature 420, 853-859]. Reciprocally, IL-1 causes AChE over-production both in PC12 cells and in the rat cortex [Li, Y. et al. (2000) J. Neurosci. 20, 149-155], suggesting a closed loop whereby ACh suppresses IL-1, ablating the induction of AChE production.
Within the mammalian spinal cord, several subsets of interneurons function in concert to translate converging cortical inputs into synchronized motoneuron activities [Noga, B. R. et al. (1995) J. Neurosci. 15, 2203-2217; Phelps, P. E. et al. (1990) J. Comp. Neural. 291, 9-26; Sherriff, F. E. and Henderson, Z. (1994) Brain Res. 634, 150-154; Perlmutter, S. I. et al. (1998) J. Neurophysiol. 80, 2475-2494; Prut, Y. and Fetz, E. E. (1999) Nature 401, 590-594]. Allostatic breakdown of this intricately controlled pathway may occur under various stressors, including glycinergic (strychnine) or cholinergic agents (succinylcholine), or under myasthenic crisis or post-anesthesia effects [Becker, C. M. et al. (1992) Neuron 8, 283-289; Millard, C. B. and Broomfield, C. A. (1995) J. Neurochem. 64, 1909-1918; Subramony, S. H. et al. (1986) Muscle Nerve 9, 64-68; Krasowski, M. D. et al. (1997) Can. J. Anaesth. 44, 525-534]. These and other acute stressors may induce massive tremor and spastic paralysis, reflecting failure of the quality control processes which presumably act to sustain cholinergic homeostasis in spinal cord motoneurons. In addition to these modulations in cholinergic neurotransmission, both injury and chemical stressors as well as organophosphate inhibitors of acetylcholinesterase (AChE) induce up-regulation of pro-inflammatory cytokines in the spinal cord (e.g. IL-1β following experimental spinal injury) [Wang, C. X. et al. (1997) Brain Res 759, 190-196; Svensson, I. et al. (2001) Neurotoxicology 22, 355-362; Dyer, S. M. et al. (2001) Toxicology 169, 177-185]. The cholinergic control over peripheral release of pro-inflammatory cytokines [Bernik, T. R. et al. (2002) J. Exp. Med. 195, 781-788; Borovikova, L. V. et al. (2000) Nature 405, 458-462; Tracey, K. J. et al. (2001) Faseb J. 15, 1575-1576] thus provoked the question whether cholinergic allostasis serves to control pro-inflammatory responses also in central nervous system (CNS) neurons.
Because spinal cord motoneurons respond to ACh, the presumed quality control process should exert regulatory effects upon cholinergic neurotransmission. As it needs to function rapidly, it likely involves short-lived molecules. Furthermore, in order to be broad-ranged, the proposed mechanism is likely to be induced under widely diverse stressors. The normally rare, stress-induced acetylcholinesterase variant AChE-R meets all of the requirements from an inducer of such response(s). AChE-R is overproduced under psychological, chemical and physical stresses [reviewed by Soreq, H. and Seidman, S. (2001) Nat. Rev. Neurosci. 2, 294-302]. A parallel stress response involves down-regulation of choline acetyltransferase (ChAT) [Kaufer, D. et al. (1998) Nature 393, 373-377] and the genomically linked vesicular acetylcholine transporter (VAChT) [Weihe, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93, 3547-3552], together limiting the production and vesicle packaging of acetylcholine while expediting its degradation. This yields down-regulation of the cholinergic hyperexcitation that is associated with many stresses. At a longer range, this stress response is associated with hypersensitivity to both agonists and antagonists of cholinergic neurotransmission [Meshorer, E. et al. (2002) Science 295, 508-512] and abnormal locomotor activities that can be ablated under antisense destruction of AChE-R mRNA [Cohen, O. et al. (2002) Mol. Psychiatry. 7, 874-885]. Finely-tuned control over AChE-R levels thus emerged as a key component of stress management by spinal cord motoneurons. AChE-R over-expression, which suppresses ACh levels, further lead to increased IL-1 production. Should this be the case, antisense suppression of AChE-R production [Brenner, T. et al. (2003) Faseb J. 17(2), 214-22] would increase ACh levels and reduce the levels of pro-inflammatory cytokines in CNS neurons.
In counterpart, parallel inflammatory responses and production of cytokines, particularly within the brain, has raised the suggestion that illness-associated alterations in memory functioning caused by medical conditions like Alzheimer's disease [Arendt, T. (2001) Neuroscience 102:723-65], multiple sclerosis [Thornton, A. E. et al. (2002) J. Int. Neuropsychol. Soc. 8:395-409], acquired immunodeficiency syndrome [Navia, B. A. et al. (1986) Ann. Neurol. 19:517-24] and infectious diseases [Capuron, L. et al. (1999) Psychol. Med. 29:291-7], are at least partly mediated by immune activation [Rachal Pugh C., et al. (2001) Neurosci. Biobehay. Rev. 25:29-41; Maier S. F. and Watkins L. R. (1998) Psychol. Rev. 105:83-107; Yirmiya R. (1997) Current Opinion in Psychiatry, 10: 470-476; Yirmiya, R. et al. (2002) Neurobiology of Learning and Memory, 78: 379-389]. Cytokine-induced memory impairments in humans, including cancer and hepatitis-C patients, as well as in experimental animals, support this notion [Capuron L. et al. (2001) Psychosom. Med. 63:376-86; Meyers C. A. (1999) Adv. Exp. Med. Biol. 461:75-81; Gibertini M. (1996) Adv. Exp. Med. Biol. 402:207-17; Oitzl M. S. et al. (1993) Brain Res. 613:160-3]. Thus, like many other stressful stimuli, which are known to affect learning and memory processes [Kim J. J. and Diamond D. M. (2002) Nat. Rev. Neurosci. 3:453-62], inflammation can cause marked alterations in memory functioning. Administration of endotoxin (lipopolysaccharide), a complex glycolipid found in the outer membrane of all gram-negative bacteria, serves to assess the cognitive consequences of the acute host response to infection in humans. Endotoxin administration induces fever, malaise and increased production and secretion of cytokines, particularly TNF-α, IL-6, IL-1 and IL-1ra and cortisol [for review see Burrell R. (1994) Circ. Shock 43:137-53], as well as proteases [Fahmi H. and Chaby R. (1994) Immunol. Invest. 23:243-58]. In healthy humans, endotoxin-induced cytokine secretion is correlated with impairments in verbal and non-verbal declarative memory functions [Reichenberg A. et al. (2001) Arch. Gen. Psychiatry 58:445-52].
Memory deficits and profound neurobehavioral and neuroendocrine symptoms were also reported to be correlated with endotoxin-induced secretion of cytokines in experimental animals [Hauss-Wegrzyniak B. et al. (2000) Neuroreport 11:1759-63; Pugh C. R. et al. (1998) Brain Behau. Immun. 12:212-29; Shaw K. N. et al. (2001) Behav. Brain Res. 124:47-54]. While these findings suggest that cytokines are involved in mediating the effects of endotoxin on memory, little is known about the neurotransmission pathways associated with these cytokine activities. The inventors initiated a search into the possibility that cholinergic processes are relevant to endotoxin responses because in the central nervous system (CNS), cholinergic responses are notably involved in several important aspects of cognitive functioning, including attention, learning and memory [for reviews see Levin E. D. and Simon B. B. (1998) Psychopharmacology (Berl) 138:217-30; Segal M. and Auerbach J. M. (1997) Life Sci. 60:1085-91]. Moreover, endotoxin decreases brain choline acetyltransferase activity [Willard L. B. et al. (1999) Neuroscience 88:193-200], similar to the effects of psychological stress [Kaufer (1998) id ibid]. In the periphery, endogenous or exogenous acetylcholine (ACh) attenuates the release of pro-inflammatory cytokines from endotoxin-stimulated human macrophages [Borovikova (2000) id ibid.; Bemik (2002) id ibid.; Tracey (2001) id ibid]. The ACh hydrolyzing enzyme acetylcholinesterase (AChE) was considered as potentially being of particular relevance to these processes because AChE controls ACh levels and since AChE inhibitors improve cognitive functions in both clinical and experimental paradigms [Palmer A. M. (2002) Trends Pharmacol. Sci. 23:426-33; Weinstock M. (1995) Neurodegeneration 4:349-56]. Moreover, AChE over-expression is triggered by acute and chronic stressful insults [Meshorer (2002) id ibid.] and induces progressive memory impairments, as was demonstrated in transgenic mice [Beeri R. et al. (1995) Curr. Biol. 5:1063-71].
Moreover, mice that overexpress both AC and AChE-R present progressive dendritic and spine loss [Beeri R. et al. (1997) J. Neurochem. 69:2441-51], as well as altered anxiety responses [Erb C. et al. (2001) J. Neurochem. 77:638-46]. Furthermore, these mice display early-onset deficits in social recognition and exaggerated responsiveness to stressful insults. These can be briefly ameliorated by conventional anticholinesterase treatment or for longer periods by an antisense oligonucleotide capable of specifically inducing the destruction of AChE-R mRNA [Cohen (2002) id ibid.], suggesting that AChE-R is the primary cause. Thus, AChE-R production may lead to both positive and negative effects on cognition.
Stressful insults induce AChE-R production in the periphery as well (e.g., in the small intestines), and failure to induce this production, in response to aversive stimuli, results in hypersensitivity to relatively mild stressors [Shapira M. et al. (2000) Hum. Mol. Genet. 9:1273-1281]. This observation raised the possibility that peripheral AChE modulations may serve as a surrogate marker of endotoxin-induced changes in cognition as well. However, in plasma, proteolytic cleavage of AChE-R leads to the appearance in the serum of a short immunopositive C-terminal peptide which facilitates the hematopoietic stress responses [Grisaru, D. et al. (2001) Mol. Med. 7, 93-105]. Hence, the inventors investigated the effects of endotoxin administration on both AChE activity and AChE-R cleavage in healthy human volunteers and explored potential correlations between these parameters, the secretion of cytokines or cortisol, and changes with time in memory functions. In addition to declarative memory, which involves consciously accessible records of facts and events through concerted functioning of hippocampal and prefrontal structures [Kim and Diamond (2002) id ibid.], the inventors assessed the effects of endotoxin and its interactions with AChE cleavage on working memory, which involves temporary storage and manipulation of information necessary for cognitive functioning [Baddeley A. (1992) Science 255:556-9], and has been shown to involve prefrontal cholinergic mechanisms [Furey M. L. et al. (2000) Science 290:2315-9].
Peripheral neurophaties are caused by altered function and structure of peripheral motor, sensory or autonomic neurons. The main causes of neuropathy are entrapment (compression), diabetes and other systemic diseases, inherited disorders, inflammatory demyelinating, ischemic, metabolic, and paraneoplastic conditions, nutritional deficiency states, and toxin-induced derangement. One example of a peripheral neuropathy is the Guillain-Barre syndrome (GBS).
GBS is an acute inflammatory polyneuropathy. It is the most common cause of acute flaccid paralysis worldwide, with an annual incidence of 0.75 to 2 in 100,000 in the general population. GBS is suspected when a patient presents with progressive motor weakness and loss of deep tendon reflexes (areflexia). Other clinical features include sensory symptoms, cranial nerve involvement, autonomic dysfunction causing pulse and blood pressure changes, and respiratory failure, which is a major cause of morbidity and mortality [Asbury and Comblath, (1990) Ann. Neurol. 27: Suppl. S21-24]. The onset of symptoms can either be acute or sub-acute, but improvement is gradual, initiating after a plateau phase of several weeks, reaching clinical recovery by 6-7 months [Group, T.I.G. (1996) Brain 119: (Pt. 6) 2053-2061]. Ventillatory support due to respiratory muscle weakness is needed in about a quarter of the patients and mortality ranges up to 13 percent [Seneviratne, U. (2000) Postgrad. Med. 76: 774-782].
In about two thirds of patients, symptoms are preceded by an antecedent infection, commonly an upper respiratory tract infection (40%) or gastroenteritis (20%) occurring 4 weeks prior to onset of disease [Group (1996) id ibid.; Rees, J. et al. (1995) N. Eng. J. Med. 333: 1374-1379]. According to this, GBS is thought to result from abnormal immune responses triggered by certain infectious agents and directed towards the peripheral nerves [Seneviratne (2000) id ibid]. Interestingly, one recent report suggests that the clinical symptoms of drug poisoning by the AChE-inhibitor rivastigmine resemble those of GBS [Lai, M. W. et al. (2005) N. Engl. J. Med. 353:3].
The diagnosis of Guillain-Barre syndrome is based on clinical presentation, which is then supported by cerebrospinal fluid (C SF) analysis demonstrating elevated protein content and normal leukocyte cell count, indicating an inflammatory reaction. Electrophysiological studies then specify the clinico-pathological type according to evidence for damage of myelin, motor or sensory axons [Asbury and Cornblath (1990) id ibid.].
Segmental demyelination, termed acute inflammatory demyelinating polyradiculoneuropathy (AIDP) is the most common type of Guillain-Barre syndrome, apparently mediated by lymphocytic and macrophage infiltration of the peripheral nerves [Griffin J. et al. (1995) Brain 118: (Pt. 3), 577-595; Honavar M. et al. (1991) Brain 114: (Pt. 3), 1245-1269; Rees (1995) id ibid.]. Demyelination is demonstrated by electrophysiological reduction of nerve conduction velocity, and subsequent remyelination is associated with recovery. In contrast to this, only minimal demyelination but prominent Wallerian-like degeneration with peri-axonal macrophage infiltration are detected in axonal degeneration types of GBS, where motor axons exclusively or motor together with sensory axons, are damaged in acute motor axonal neuropathy (AMAN) [McKhann G. et al. (1993) Ann. Neural. 33: 333-342] and acute motor sensory axonal neuropathy (AMSAN) [Griffin (1995) id ibid.], respectively. Accordingly, the electrophysiological features in these cases are reduced compound muscle action potential (CMAP) amplitude, and additionally, reduced sensory nerve action potentials in AMSAN, but preserved conduction velocity, indicating axonal dysfunction without demyelination. Both axonal neuropathies are characterized by rapidly progressive weakness, often with respiratory failure, but although AMAN patients usually exhibit good recovery [McKhann (1993) id ibid.], the recovery of AMSAN patients is generally slow and incomplete, considered to be the most severe form of GBS (Brown and Feasby (1984) Brain 107: (Pt. 1) 219-239].
Axonal degeneration types of GBS are often preceded by infection with Campylobacter jejuni (Cj), which is associated with a slow recovery, and severe residual disability [Rees (1995) id ibid]. There are several serotypes of Cj, and the one most often isolated from GBS patients belongs to Penner serotype 19 (0:19) (Saida, T. et al. (1997) J. Infect. Dis. 176: Suppl. 2, S129-134]. The lipopolysaccharides (LPS) of Cj share ganglioside-like epitopes with ganglioside-surface molecules of peripheral nerves, and patients with GBS have anti-ganglioside antibodies, suggesting that “molecular mimicry” is the immunopathogenic mechanism of injury to the peripheral nerve fibers [Sheikh, K. et al., (1998) Ann. N.Y. Acad. Sci. 845: 307-321; Yuki N. et al., (1993) J. Exp. Med. 178: 1771-1775]. Nevertheless, although Cj-0:19 serotype is significantly associated with elevated anti-ganglioside antibody titers in the sera of the patients, no significant correlation was found between the presence of these antibodies and the clinical pattern of GBS [Nishimura M. et al. (1997) J. Neurol. Sci. 153: 91-99]. This therefore indicates that additional factors may determine the axonal damage or disfunction following the apparently antibody-mediated nerve-surface injury. In agreement with this, the currently accepted treatments of GBS is intravenous immunoglobulin administration or plasma exchange (plasmapheresis), which act through suppression or removal of auto-antibodies, both which have been found to be equally beneficial [Seneviratne (2000) id ibid]. Nevertheless, several authors reported a rapid resolution of nerve conduction blocks following plasmapheresis, which could not be explained by remyelination or axonal regeneration [Kuwabara S. et al., (1999) Muscle Nerve 22: 840-845; Suzuki and Choi, (1991) Acta. Neuropathol. (Berl) 82: 93-101]. This suggests a possible role for a humoral factor in the pathogenesis of the disease, causing physiological conduction abnormalities that may facilitate the destructive process.
Administration of LPS to humans is known to increase production and secretion of cytokines and cortisol [Burrell R. (1994) Circ. Shock 43: 137-153]. In addition to this, LPS decreases the activity of brain choline acetyltransferase [Willard L. et al. (1999) Neuroscience 88: 193-200], similar to the effects of psychological stress [Kaufer (1998) id ibid.], reducing production of acetylcholine (ACh). In the periphery, ACh attenuates the release of pro-inflammatory cytokines from LPS-stimulated human macrophages [Bemik, T. et al. (2002) J. Exp. Med. 195: 781-788; Borovikova L. et al. (2000) Nature 405: 458-462; Tracey K. et al. (2001) Faseb. J. 15: 1575-1576]. AChE is therefore considered as potentially being of particular relevance to these processes because AChE controls ACh levels. Acute and chronic stressful insults trigger transcriptional activation of AChE gene expression, which leads to accumulation of the normally rare, AChE-R splice variant [Soreq (2001) id ibid]. The AChE-R excess reduces the stress-induced cholinergic hyperexcitation in the CNS [Kaufer (1998) id ibid]. In the periphery (e.g., in the small intestines), failure to induce this production in response to aversive stimuli results in hypersensitivity to relatively mild stressors [Shapira (2000) id ibid]. In plasma, proteolytic cleavage of AChE-R leads to the appearance of its distinct short C-terminal peptide (AChE-R Peptide; ARP) which accumulates following Salmonella-LPS endotoxin administration to humans [Cohen O. et al. (2003) J. Mol. Neurosci. 21: 199-212], and facilitates the hematopoietic stress responses [Grisaru (2001) id ibid]. The inventors hence sought to examine the involvement of AChE-R and ARP in induction of functional conduction abnormalities in the sciatic nerve.
The role of cholinergic mechanisms in learning and memory, the involvement of AChE-R in stress responses, the suppression by ACh of pro-inflammatory cytokines production, the effects of endotoxin on memory functions, and the potential involvement of AChE-R in nerve conduction block, suggested involvement of AChE-R in inflammatory associated processes which could thus potentially be suppressed by an inhibitor of AChE-R expression.
The prospect of therapeutic agents of exquisite specificity and action at very low concentration has stimulated the development of antisense oligonucleotides (AS-ON) targeted against a variety of mRNAs. Major problems remain access to the RNA processing machinery of the cell, potential differences between specific cell types and the mode of chemical protection employed. When the cell of interest is within the CNS, the problem of access is compounded by the presence of the blood-brain barrier [Tavitian, B. et al. (1998) Nat. Med. 4, 467-471]. Nevertheless, some attempts have been successful even in primates [Kasuya, E. et al. (1998) Regul. Pept. 75-76, 319-325; Mizuno, M. et al. (2000) Endocrinology 141, 1772-1779]. The inventors have previously demonstrated antisense suppression of the stress-induced AChE-R mRNA, enabling retrieval of normal cellular and physiological functions following stress-induced changes in cultured rat and human cells [Galyam, N. et al. (2001) Antisense Nucleic Acid Drug Dev. 11, 51-57; Grisaru, D. et al. (2001) id ibid.] and in live mice [Cohen et al. (2002) id ibid.; Shohami, E. et al., (2000) J. Mol. Med. 78, 228-236] and rats [Brenner, T. et al., (2003) id ibid]. While the tested consequences in all of these studies were limited to direct measurement of the target protein and mRNA, the working hypothesis predicted additional, anti-inflammatory effects for antisense retrieval of cholinergic balance. Here, the inventors report the outcome of experiments aimed at addressing the stress-induced overproduction and selective AS-ON retrieval of normal AChE-R levels under injection stress in cynomolgus monkeys. The findings demonstrate differential susceptibility of specific neuron types to AS-ON responses, as well as concomitant suppression of IL-1β and IL-6 following the retrieval of cholinergic balance in spinal cord neurons. The present inventors have previously found that antisense oligonucleotides against the common coding region of AChE are useful for suppressing AChE-R production [see WO 98/26062]. In particular, the inventors have shown the use of an antisense oligonucleotide against the AChE sequence for the treatment of myasthenia gravis [WO 03/002739 and US 2003/0216344].
Various diseases are associated with chronic inflammation of the gastrointestinal tract. These diseases include inflammatory bowel diseases (e.g. ulcerative colitis and Crohn's disease), irritable bowel syndrome, ileitis, chronic inflammatory intestinal disease and celiac.
Inflammatory Bowel Disease (IBD) is a chronic, recurring-remitting immune response and inflammation of the gastrointestinal tract. The two most common conditions of IBD are ulcerative colitis (UC) and Crohn's disease. Currently, the annual incidence of IBD ranges from 1 to 10 cases per 100,000 and the prevalence ranges from 10 to 70 per 100,000 people.
Crohn's disease (also known as Crohn syndrome and regional enteritis), is a type of IBD that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms. It primarily causes abdominal pain, diarrhea (which may be bloody), vomiting or weight loss, but may also cause complications outside the gastrointestinal tract such as skin rashes, arthritis, inflammation of the eye, tiredness, and lack of concentration.
Ulcerative colitis is a lifelong illness that has a profound health-related, emotional and social impact on affected patients. Ulcerative colitis affects the lining of the large intestine (colon) and rectum and may affect any age group, although there are peaks at ages 15-30 and then again at ages 50-70. Similar to the case of Crohn's disease, the symptoms of ulcerative colitis include abdominal pain and cramping, blood in the stools, diarrhea, fever, rectal pain and weight loss.
Current treatment of IBD is aimed at reducing its symptoms, pushing patients into remission and maintaining them at that state. Treatment can be broadly divided into anti-inflammatory (e.g., sulfasalazine, 5-aminosalicylic acid), immunosuppressant (e.g., azathioprine, 6-mercaptopurine) and biological drugs (e.g. Remicade and Humira). Anti-inflammatory drugs are usually the first-line treatment. Corticosteroids or TNF blockers (e.g., infliximab, adalimumab, certolizumab) may be an alternative or additional treatment for patients with moderate to severe IBD who do not respond to the first-line treatment, in order to reduce inflammation. Unfortunately, however, all these treatments are associated with significant adverse effects. Therefore, there remains a significant unmet medical need for novel efficacious treatments with a favorable safety profile.
Based on the inventors' herein described results, the present invention provides a novel use for an antisense oligonucleotide directed against the AChE mRNA sequence, as a new anti-inflammatory agent, specifically for the treatment of subjects afflicted with gastrointestinal inflammatory disorders.
Other purposes and advantages of the invention will become apparent as the description proceeds.